The present invention relates generally to the field of recombinant DNA technology and, more particularly, to improved methods for producing amplified heterogeneous populations of polynucleotides from limited quantities of DNA or RNA or other nucleic acids. The invention provides compositions and methods for a chain reaction amplification of a target polynucleotide species using a thermostable polymerase or other suitable polynucleotide polymerase compatible with the method.
Selective amplification of polynucleotides represents a major research goal of molecular biology, with particular importance in diagnostic and forensic applications, as well as for general manipulations of genetic materials and laboratory reagents.
The polymerase chain reaction (PCR) is a method by which a specific polynucleotide sequence can be amplified in vitro. PCR is an extremely powerful technique for amplifying specific polynucleotide sequences, including genomic DNA, single-stranded cDNA, and mRNA among others. As described in U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,800,159 (which are incorporated herein by reference), PCR typically comprises treating separate complementary strands of a target nucleic acid with two oligonucleotide primers to form complementary primer extension products on both strands that act as templates for synthesizing copies of the desired nucleic acid sequences. By repeating the separation and synthesis steps in an automated system, essentially exponential duplication of the target sequences can be achieved.
A number of variations of the basic PCR methodology have been described. U.S. Pat. No. 5,066,584 discloses a method wherein single stranded DNA can be generated by the polymerase chain reaction using two oligonucleotide primers, one present in a limiting concentration. U.S. Pat. No. 5,340,728 discloses an improved method for performing a nested polymerase chain reaction (PCR) amplification of a targeted piece of DNA, wherein by controlling the annealing times and concentration of both the outer and the inner set of primers according to the method disclosed, highly specific and efficient amplification of a targeted piece of DNA can be achieved without depletion or removal of the outer primers from the reaction mixture vessel. U.S. Pat. No. 5,286,632 discloses recombination PCR (RPCR) wherein PCR is used with at least two primer species to add double-stranded homologous ends to DNA such that the homologous ends undergo in vivo recombination following transfection of host cells.
Horton et al. (1989) Gene 77: 61, discloses a method for making chimeric genes using PCR to generate overlapping homologous regions. In the Horton method, fragments of different genes that are to form the chimeric gene are generated in separate polymerase chain reactions. The primers used in these separate reactions are designed so that the ends of the different products of the separate reactions contain complementary sequences. When these separately produced PCR products are mixed, denatured and reannealed, the strands having matching sequences at their 3xe2x80x2-ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are spliced together to form the chimeric gene.
Silver and Keerikatte (1989) J. Virol. 63: 1924 describe another variation of the standard PCR approach (which requires oligonucleotide primers complementary to both ends of the segment to be amplified) to allow amplification of DNA flanked on only one side by a region of known DNA sequence. This technique requires the presence of a known restriction site within the known DNA sequence and a similar site within the unknown flanking DNA sequence which is to be amplified. After restriction and recircularization, the recircularized fragment is restricted at an unique site between the two primers and the resulting linearized fragment is used as a template for PCR amplification.
Triglia et al. (1988) Nucl.Acids Res. 16: 8186, describe an approach which requires the inversion of the sequence of interest by circularization and re-opening at a site distinct from the one of interest, and is called xe2x80x9cinverted PCR.xe2x80x9d A fragment is first created in which two unknown sequences flank on either side a region of known DNA sequence. The fragment is then circularized and cleaved with an unique restriction endonuclease which only cuts within the known DNA sequence creating a new fragment containing all of the DNA of the original fragment but which is then inverted with regions of known sequence flanking the region of unknown sequence. This fragment is then utilized as a PCR substrate to amplify the unknown sequence.
Vallette et al. (1989) Nucl.Acids Res. 17: 723 disclose using PCR in a specific approach which involves using a supercoiled plasmid DNA as a template for PCR and a primer bearing a mutated sequence which is incorporated into the amplified product. Using this method, DNA sequences may be inserted only at the 5xe2x80x2-end of the DNA molecule which one wishes to alter. Mole et al. (1989) Nucl.Acids Res. 17: 3319, used PCR to create deletions within existing expression plasmids. However, PCR was performed around the entire plasmid (containing the fragment to be deleted) from primers whose 5xe2x80x2-ends defined the region to be deleted. Self-ligation of the PCR product recircularized the plasmid.
U.S. Pat. No. 5,279,952 discloses a method for using PCR to generate mutations (e.g., deletions) and chimeric genes by forming head-to-tail concatemers of a known starting sequence and employing at least two PCR primers to amplify a DNA segment which is altered as compared to the known starting sequence.
Jones and Howard (1990) BioTechniques 8: 178, report a site-specific mutagenesis method using PCR, termed recombinant circle PCR (RCPCR). In RCPCR, separate PCR amplifications (typically two) of a known polynucleotide generate products that, when combined, denatured, and annealed, form double-stranded DNA with discrete, cohesive single-stranded ends designed so that they may anneal and form circles of DNA.
Oliner et al. (1993) Nucl. Acids. Res. 21: 5192, report a method for engineering PCR products to contain terminal sequences identical to sequences at the two ends of a linearized vector such that co-transfection of the PCR product and linearized vector into a recombination-competent host cell results in formation of a covalently linked vector containing the PCR product, thus avoiding the need for in vitro ligation.
In spite of such recent advances, including PCR and its various modifications noted above, there exists a need for improved methods of identifying and cloning polynucleotides, for accurate in vitro amplification of selected polynucleotides, and for facile assembly of polynucleotides from a mixture of component oligonucleotides or polynucleotides without necessitating the use of DNA ligase. In particular, there is a need for a PCR amplification method which can be performed with (1) only a single primer species, or (2) with multiple overlapping polynucleotide fragments (or oligonucleotides) in the absence of a conventional PCR primer, and which can result in formation of an amplified product which can be a concatemer and/or which can be a covalently-closed circle. The present invention fulfills these and other needs.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. All publications cited are incorporated herein by reference.
A basis of the present invention is the use of polymerase-mediated chain extension, such as for example PCR, in combination with at least two polynucleotides having complementary ends which can anneal whereby at least one of said polynucleotides has a free 3xe2x80x2-hydroxyl capable of polynucleotide chain elongation by a DNA polymerase, such as a thermostable polymerase (e.g., Thermus apuaticus (Taq) polymerase or Thermococcus litoralis (Vent(trademark), New England Biolabs) polymerase or TthI polymerase (Perkin-Elmer). Although the method can be practiced using PCR, in some embodiments either a single primer species or no primer whatsoever is required, and hence PCR is not a necessary component of the general method.
In one embodiment, a target polynucleotide is contacted with a xe2x80x9cbivalent primerxe2x80x9d typically comprising an oligonucleotide having two regions of complementarity to the target polynucleotide: (1) a first portion which is in the 5xe2x80x2 portion of the primer and which is substantially complementary to a sequence in the 5xe2x80x2-portion of the sequence to be amplified (target sequence) in the target polynucleotide, and (2) a second portion which is in the 3xe2x80x2 portion of the primer and which is substantially complementary to a sequence in the 3xe2x80x2-portion of the sequence to be amplified (target sequence) in the target polynucleotide. The contacting is performed under conditions suitable for hybridization of the bivalent primer to the target polynucleotide, most often following thermal denaturation of the target polynucleotide if it is initially present as a double-stranded form. The target polynucleotide may be substantially homogeneous or may be present in a mixture of polynucleotide species (e.g., in a genome, biological sample, or mixture of synthetic polynucleotides). Subsequent or concomitant with the contacting of the bivalent primer to the target polynucleotide, a polynucleotide polymerase, such as a thermostable DNA polymerase, catalyzes, under suitable reaction conditions, polynucleotide synthesis (chain elongation) primed from the 3xe2x80x2-hydroxyl of the annealed bivalent primer to form a strand complementary to the target sequence, thereby forming a nascent complementary strand. Following completion of the nascent complementary strand spanning the target sequence, the target polynucleotide and the nascent strand are denatured, typically by elevation of temperature, and allowed to reanneal, typically by reduction of temperature, with another molecule of the bivalent primer species or with a complementary strand of a target polynucleotide or an amplified copy thereof. The denatured nascent strand species following the first elongation cycle will contain a copy of the target sequence and has a terminal repeat of its 5xe2x80x2-terminal sequence at the 3xe2x80x2 terminus, resulting from the bivalent primer, and wherein the terminal repeat is of sufficient length to support annealing under PCR conditions to an overlapping complementary strand in a head-to-tail arrangement (see, FIG. 1). Following reannealing, the described polymerase elongation/denaturation/reannealing cycle is repeated from 1 to about 100 times as desired, resulting in formation of amplified product which comprises head-to-tail concatemers of the target sequence. The concatemers typically increase in length as the number of amplification cycles increase and as the amount of bivalent primer decreases. Following amplification forming concatameric head-to-tail repeats of the target sequence, the concatemer(s) can optionally be resolved, such as (1) by cleaving with a restriction endonuclease which cuts within (or at the termini of) the concatemeric unit(s), (2) by homologous recombination between concatemer units to form covalently closed circles, or (3) by cleavage with a restriction endonuclease followed by ligation with DNA ligase to form covalently closed circles and/or by direct transformation into host cells for in vivo ligation.
Often, a target polynucleotide sequence which is amplified as described above will form amplification intermediates in the form of cyclized DNA or spiral DNA (see, FIG. 2), as a result of the 3xe2x80x2 terminus of an overlapped nascent strand annealing to the 3xe2x80x2 terminus of an overlapped complementary strand forming a cyclized structure similar to a gapped circle; the cyclized structure has a strand with an extendable 3xe2x80x2-hydroxyl which can be extended with a DNA polymerase substantially lacking exonuclease activity (e.g., a thermostable polymerase such as Vent(exoxe2x88x92)(trademark) or Klenow fragment, etc.) in a rolling circle format whereby the leading terminus of the nascent strand continually displaces the lagging portion of the nascent strand (see, FIG. 2) producing a concatemeric single strand emanating from the rolling circle intermediate. Most often, such rolling circle intermediates will form under dilute conditions more favorable to intramolecular cyclization of overlapped strands. Once a rolling circle intermediate is established, the template need not be denatured in order to continue amplification of the target sequence as in conventional PCR, thus avoiding the necessity of multiple thermal cycles of PCR to denature template (and the resultant time loss needed for heating and cooling). Often, however, the template is repeatedly denatured, annealed, and extended with polymerase in the presence of ribonucleotide or deoxyribonucleotides under suitable reaction conditions.
Furthermore, whether the method generates a rolling circle intermediate or linear concatemers, an advantage of the method is that it requires a substantially reduced amount of primer (bivalent primer) as compared to conventional PCR, since following the initial cycle(s) an increasing percentage of the priming of nascent strand synthesis is primed from 3xe2x80x2-hydroxyl groups of the amplified strands, rather than from the oligonucleotide primer(s). In the case of a rolling circle intermediate, theoretically only a single bivalent primer molecule is necessary to generate the rolling circle which then can produce multiple concatenated copies by rolling circle-style polymerase catalysis using a polymerase capable of strand displacement of the lagging edge of the nascent strand as replication proceeds around the cyclized template.
In an embodiment, a product polynucleotide is assembled from a plurality of component polynucleotides by formation of overlapped strands of alternating polarity and having substantially complementary termini (see, FIG. 3). This method employs a series of overlapping substantially complementary termini to determine the linear order of component sequences in the final product. Concomitant with or subsequent to formation of the overlapped strands of the component polynucleotides in a reaction, a polynucleotide polymerase (e.g., a thermostable DNA polymerase) under suitable reaction conditions catalyzes strand elongation from the 3xe2x80x2-hydroxyl portions of the overlapped (annealed) joints, filling in the portion between joints and processively displacing or processively degrading exonucleolytically the 5xe2x80x2 termini of downstream component strands of the same polarity as the nascent strand elongates. After a cycle of chain elongation forming substantially double-stranded polynucleotides, the reaction conditions are altered (typically by increasing the temperature) to effect denaturation of the double-stranded polynucleotides, followed by altering the reaction conditions to permit reannealing of complementary strands or portions thereof (i.e., overlapping termini) to form molecules having overlapped termini (joints), and chain elongation by a polynucleotide polymerase under suitable reaction conditions catalyzes strand elongation from the 3xe2x80x2-hydroxyl portions of the overlapped (annealed) joints, as in the first cycle. One to about 100 cycles of denaturation/annealing/polymerization can be performed to generate a product comprising the component polynucleotide sequences covalently linked in linear order according to the order of the overlapping joints. In this embodiment, a product polynucleotide can be constructed from a plurality of smaller component polynucleotides (typically oligonucleotides) and enables assembly of a variety of products with alternate substitutable polynucleotide components at a given position serving as structural xe2x80x9callelesxe2x80x9d (see, FIG. 4). The component polynucleotides are often provided in single-strand form, but may initially be present in double-strand form and be denatured (typically by elevated temperature) for the assembly of the product by PCR amplification. Substantially any type of product polynucleotide can be assembled in this way, including cloning and expression vectors, viral genomes, gene therapy vectors, genes (including chimeric genes), polynucleotides encoding peptide libraries, protein libraries, vector libraries, viral libraries, and the like. In a variation, one or more of the component polynucleotides represents a site-directed mutation or variable-sequence kernel. In a variation, PCR employing a low-fidelity polymerase is used to introduce additional sequence variation into the product polynucleotide(s) during amplification cycles. The method can be used to produce a library of sequence-variant product polynucleotides, if desired.
In an embodiment of the invention, very long distance PCR is provided, wherein PCR or other suitable amplification method is used to generate, in a single reaction or in parallel reactions which are subsequently pooled, a set of overlapping large DNA fragments which can be denatured and annealed to form very large (e.g., greater than 25 to 50 kilobases) DNA structures composed of overlapped single strands of DNA having alternating polarity with each overlapped joint providing an extendable 3xe2x80x2-hydroxyl group for forming phosphodiester bonds catalyzed by a polynucleotide polymerase in the presence of free ribonucleotide or deoxyribonucleotides. Typically, the method comprises forming at least three overlapping polynucleotides, wherein the 3xe2x80x2 terminus of a first single-stranded polynucleotide is substantially complementary to the 3xe2x80x2 terminus of a second single-stranded polynucleotide of the opposite polarity, and wherein the 5xe2x80x2 terminus of said second single-stranded polynucleotide is substantially complementary to the 3xe2x80x2 terminus of a third single-stranded polynucleotide having polarity identical to said first single-stranded polynucleotide, thereby generating an overlapped structure capable of chain elongation by a suitable polymerase to generate a double-stranded product spanning the three initial overlapped polynucleotides. With such a method, polynucleotides of 50 kb to 100 kb or more can be generated by a facile amplification method capable of generating amplification products much longer than is possible with conventional long-range PCR methods. The method can comprise parallel processing PCR reactions, wherein a plurality of primer sets are employed in a single reaction or multiple reactions which are subsequently pooled, each primer sets priming the PCR amplification of a polynucleotide sequence which comprises terminal sequences which are complementary to terminal sequences in at least one other amplification product produced by a different primer set, thus generating a set of overlapping PCR products with which a large product spanning the entire set of PCR products is generated by end-complementary polymerase reaction.
In some embodiments of the invention, the polynucleotides product(s) generated thereby are labelled, such as with radioisotopic, biotinyl, or fluorescent label moieties, by incorporation of labelled ribonucleotide or deoxyribonucleotides or the like into nascent polynucleotide by polymerase-mediated catalysis.
The invention also provides kits comprising a bivalent primer polynucleotide and/or a plurality of component polynucleotides and instructions for use describing the present end-complementary amplification method disclosed herein. Frequently, a polynucleotide polymerase, such as a thermostable DNA polymerase (Taq or Vent(trademark) polymerase) is also present in the kit. Optionally, one or more target polynucleotides may be provided in the kit, such as for calibration and/or for use as a positive control to verify correct performance of the kit.
In an embodiment, the invention provides a method termed continuous multiplex amplification which affords amplification of a plurality of initially unlinked polynucleotide species at substantially comparable amplification rates by forming a linked amplification product wherein the plurality of initially unlinked polynucleotide sequences are linked by end-complementary amplification. An amplification unit, termed an amplicon, comprising at least one copy of each member of the plurality of initially unlinked polynucleotide species is formed by one or more cycles of end-complementary amplification. From one to about 100, typically three to 35, amplification cycles can be conducted and result in formation of a population of linked amplification products, which can comprise concatemers of said amplicon. The amplification products can be linear or circular, as desired, based on appropriate selection of the bivalent primers. In a variation, the amplification product is cleaved with a nucleolytic agent, such as a restriction enzyme which cuts at least one restriction site present in the amplicon, DNase, nuclease S1, bleomycin, ionizing radiation, or the like or by other suitable cleaving means.
A further understanding of the nature and advantages of the invention will become apparent by reference to the remaining portions of the specification and drawings.